dsRNAs targeting LPAs and their use

Abstract

This invention relates to LPA-targeting dsRNA and its use. It also relates to LPA-targeting double-stranded ribonucleic acid (dsRNA) and methods for its preparation. Furthermore, it relates to pharmaceutical compositions, cells, or reagent kits containing the dsRNA. The dsRNA can interfere with LPA expression and prevent and / or treat related diseases.

Description

[Technical Field]

[0001] This disclosure claims priority to Chinese Patent Application No. 202310628559.8 filed on 31 May 2023 and to Chinese Patent Application No. 202311386740.9 filed on 25 October 2023, and this disclosure refers to the full text of the aforementioned Chinese Patent Applications.

[0002] This disclosure belongs to the biopharmaceutical field and specifically relates to dsRNAs targeting LPAs, their use, and methods for preparation. [Background technology]

[0003] First discovered in 1963 by Norwegian geneticist Berg, lipoprotein (a) [Lp(a)] was identified as a unique lipoprotein (Berg KA new serum type system in man-the Lp system. Acta Pathol Microbiol Scand 1963, 59:369-82). Lp(a) consists of two parts: a lipid portion, which is mainly located in the core and is an LDL-like microparticle, and a protein portion, which is located in the periphery and is linked to apolipoprotein (a) [apo(a)] and apoB100 by disulfide bonds. apo(a) is mainly expressed in the liver, and its expression is limited to humans and non-human primates. It is characterized by the presence of three domains (Kringle) in a tricyclic structure stabilized by internal disulfide bonds. In human lineages, amplification and differentiation of the Kringle IV domain in apo(a) result in 10 different types of KIV domains. Further amplification of Kringle IV type 2 (KIV-2) leads to intra-alleletic copy number variation (CNV) (1-40 copies), while the other Kringle IV coding domains (KIV-1 and KIV-3-KIV-10) exist only as single copies (Schmidt K, Noureen A, Kronenberg F, et al. Structure, Function, and Genetics of Lipoprotein(a)[J]. Journal of Lipid Research, 2016, 57(8):1339). Since all Kringle domains are transcribed and translated, KIV-2 CNVs result in size polymorphism of the encoded apo(a), and their expression is inversely proportional to the number of KIV-2 domains present. When the KIV-2 copy number is relatively low, the plasma content of Lp(a) increases significantly.

[0004] Patients with elevated Lp(a) levels have a 2-3 times higher risk of cardiovascular events compared to the normal population, including atherosclerotic cardiovascular disease, lower extremity arterial disease, and aortic stenosis (EnAs EA, Varkey B, Dharmarajan TS, et al. Lipoprotein(a): An independent, genetic, and causal factor for cardiovascular disease and acute myocardial infarction[J]. Indian Heart Journal, 2019, 71(2).). Lp(a) can lead to vascular occlusion or promote thrombus formation by promoting thrombus formation in plaque rupture or turbulence in vascular stenosis, as apo(a) has been shown to inhibit fibrinolysis in vitro. On the other hand, LDL-like particles can promote intimal cholesterol deposition, inflammation, or oxidized phospholipids, leading to atherosclerotic stenosis or aortic stenosis. These two mechanisms can contribute to poor atherosclerotic cardiovascular disease (ASCVD) (Albert Youngwoo Jang, Seung Hwan Han, Il Suk Sohn, et al. Lipoprotein(a) and Cardiovascular Diseases[J]. Circulation Journal, 2020, 84:867~874). However, even at very high levels of Lp(a), its cholesterol content is lower than the conventional LDL limit, so the pathogenicity of the LDL-like particle portion may be relatively low.

[0005] In 2016, the Chinese guidelines for the prevention and treatment of adult dyslipidemia defined Lp(a) abnormality as >30 mg / dl, and based on this criterion, approximately 30% of patients in China with a history of cardiovascular events have Lp(a) abnormality. In 2019, the American Lipid Society recommended that Lp(a) ≥50 mg / dl is considered elevated, and based on this criterion, elevated Lp(a) levels exist in 20% of the world's population. Although elevated Lp(a) levels are commonly observed, there are no targeted therapies, and no drugs targeting Lp(a) reduction have ever been approved for clinical use. The Lp(a) protein is structurally similar to many lipoproteins and is not easily targeted directly by small and large molecule drugs. However, mRNA transcribed from the Lp(a) gene has high proprietaryity, as it can be specifically degraded by the siRNA post-transcriptional regulatory mechanism, further inhibiting Lp(a) expression. Therefore, siRNA targeting the apo(a) gene (LPA) is designed to reduce serum Lp(a) levels and further reduce cardiovascular adverse events by weakening its expression. [Overview of the project]

[0006] This disclosure provides a double-stranded ribonucleic acid comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region (preferably the sense strand and the antisense strand are inversely complementary), and the double-stranded ribonucleic acid targets an LPA gene or its expression product.

[0007] In some embodiments, the sense strand and / or antisense strand of the dsRNA contains at least one modified nucleotide.

[0008] This disclosure relates to a double-stranded ribonucleic acid (dsRNA) that targets LPA, comprising a sense strand and an antisense strand forming a double-stranded region, wherein The bare nucleotide sequence of the sense strand described above contains at least 17 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by 3 or fewer nucleotides, and The bare nucleotide sequence of the antisense strand described above contains at least 19 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by three or fewer nucleotides, where, in the direction from the 5' end to the 3' end, The sense strand described above consists of nucleotides modified with 2'-fluoro groups at positions 7, 8, and 9, and nucleotides modified with 2'-methoxy groups at the remaining positions. The antisense chain described above consists of nucleotides modified with 2'-fluoro groups at positions 2 and 14, nucleotides independently modified with 2'-methoxy groups or 2'-fluoro groups at positions 4, 6, 10, 12, 16, and 18, and nucleotides modified with 2'-methoxy groups at the remaining positions. The present invention provides a dsRNA in which the number of 2'-fluoromodified nucleotides in the antisense strand is 2 to 7 (for example, 2, 3, 4, 5, 6, or 7).

[0009] In some embodiments, the bare nucleotide sequence of the sense strand of the dsRNA includes or is selected from the nucleotide sequence shown in SEQ ID NO: 1, and the bare nucleotide sequence of the antisense strand includes or is selected from the nucleotide sequence shown in SEQ ID NO: 2, where, in the direction from the 5' end to the 3' end, The sense strand described above consists of nucleotides modified with 2'-fluoro groups at positions 7, 8, and 9, and nucleotides modified with 2'-methoxy groups at the remaining positions. The antisense chain described above consists of nucleotides modified with 2'-fluoro groups at positions 2 and 14, nucleotides independently modified with 2'-methoxy groups or 2'-fluoro groups at positions 4, 6, 10, 12, 16, and 18, and nucleotides modified with 2'-methoxy groups at the remaining positions. The number of 2'-fluoromodified nucleotides in the antisense strand described above is between 2 and 7 (for example, 2, 3, 4, 5, 6, or 7).

[0010] In some embodiments, the sense chain and antisense chain are The sense strand contains 5'-GCUCCUUAUUGUUAUACGA-3', The antisense chain contains 5'-UCGUAUAACAAUAAGGAGCUG-3'.

[0011] In some other embodiments, the sense chain and antisense chain are: The bare nucleotide sequence of the sense strand is 5'-GCUCCUUAUUGUUAUACGA-3', The bare nucleotide sequence of the antisense strand is 5'-UCGUAUAACAAUAAGGAGCUG-3', A "bare sequence" refers to an unmodified nucleotide sequence.

[0012] In some embodiments, the nucleotide at position 7 of the 5' end of the antisense strand of the dsRNA is a modified nucleotide, where the modified nucleotide is a nucleotide modified with a 2'-methoxy group.

[0013] In some embodiments, the nucleotide at position 7 of the 5' end of the antisense strand of the dsRNA is a modified nucleotide, where the modified nucleotide includes the chemical modifications shown in formulas (I), (I-1), (I-2) or pharmaceutically acceptable salts thereof. [ka] Here, B is the same base as when the nucleotide at position 7 of the 5' end of the antisense strand described above is unmodified, and in some specific embodiments, B is adenine.

[0014] In some embodiments, the chemical modifications shown in formulas (I'), (I'-1), and (I'-2) are [ka] Selected from, Here, M is O or S, and B is the same base as when the nucleotide at position 7 of the 5' end of the antisense strand is unmodified, and in some specific embodiments, B is adenine.

[0015] In some specific embodiments, M is S. In some specific embodiments, M is O.

[0016] In some embodiments, the nucleotide at position 1 of the 5' end of the antisense strand of the dsRNA is a modified nucleotide, where the modified nucleotide is a nucleotide modified with a 2'-methoxy group.

[0017] In some embodiments, the nucleotide at position 1 of the 5' end of the antisense strand of the dsRNA is a modified nucleotide, where the modified nucleotide is a nucleotide containing the chemical modification shown in formula (IV). [ka] Here, R A1 , R A2 Each is independently selected from hydrogen or deuterium. M1 and M2 are each independently selected from -SH or -OH. B is selected from a base, hydrogen, and deuterium, R A3 The group is selected from hydrogen, deuterium, hydroxyl group, halogen, alkyl group (e.g., C1, C2, C3, C4, C5, C6 alkyl groups, including but not limited to methyl, ethyl, and isopropionic groups), and alkoxy group (e.g., C1 alkoxy group, C2 alkoxy group, C3 alkoxy group, C4 alkoxy group, C5 alkoxy group, C6 alkoxy group, including but not limited to methoxy, ethoxy, propoxy, and isopropoxy groups), and the above hydroxyl group, alkyl group, and alkoxy group are each optionally substituted with one or more deuterium atoms, R A4is selected from hydrogen, deuterium, and alkyl groups (for example, C1, C2, C3, C4, C5, C6 alkyl groups, including but not limited to methyl group, ethyl group, and isopropyl group), and each of the above alkyl groups is optionally substituted with one or more deuteriums, with the condition that formula (IV) contains at least one deuterium.

[0018] In some embodiments, R A3 is selected from hydrogen and deuterium.

[0019] In some other embodiments, R A3 is selected from halogens (for example, fluorine, chlorine, bromine).

[0020] In some other embodiments, R A3 is selected from alkyl groups (for example, C1, C2, C3, C4, C5, C6 alkyl groups, including but not limited to methyl group, ethyl group, and isopropyl group), and each of the above alkyl groups is optionally substituted with one or more deuteriums.

[0021] In some other embodiments, R A3 is selected from alkoxy groups (for example, C1 alkoxy group, C2 alkoxy group, C3 alkoxy group, C4 alkoxy group, C5 alkoxy group, C6 alkoxy group, including but not limited to methoxy group, ethoxy group, propoxy group, and isopropoxy group), and each of the above alkoxy groups is optionally substituted with one or more deuteriums.

[0022] In some embodiments, the 5'-terminal chemical modification shown in formula (IV) is

Chemical formula

[0023] In some embodiments, R A5 , R A6 , R A7 These are all deuterium.

[0024] In some embodiments, R A5 It is deuterium, and R A6 , R A7 It is hydrogen.

[0025] In some embodiments, R A5 , R A6 It is deuterium, and R A7 It is hydrogen.

[0026] In some embodiments, R A1 is hydrogen, R A2 It is deuterium.

[0027] In some embodiments, R A1 and R A2 It is deuterium.

[0028] In some embodiments, R A1 and R A2 It is hydrogen.

[0029] In some embodiments, the 5'-terminus chemical modification shown in formula (IV) is [ka] Selected from JPEG2026520380000006.jpg83151, B is selected from a base or hydrogen.

[0030] In some embodiments, B is selected from a base, and in some specific embodiments, B is selected from adenine, guanine, cytosine, uracil, or thymine.

[0031] In some specific embodiments, B is a base at a position corresponding to a modified nucleotide in the antisense strand.

[0032] In some specific embodiments, B is selected from uracil.

[0033] In some embodiments, the 5'-terminus chemical modification shown in formula (IV) is [ka] The structure is selected from those in which uracil is substituted with adenine, guanine, cytosine, or thymine.

[0034] In some embodiments, following the direction from the 5' end to the 3' end, the nucleotide at position 1 of the 5' end of the antisense strand of the dsRNA is the 5' end chemically modified nucleotide shown in formula (IV). In some specific embodiments, the 5' end chemically modified nucleotide shown in formula (IV) is [ka] Therefore, B above is the base at the position corresponding to the nucleotide at position 1 of the 5' end of the antisense strand.

[0035] In some embodiments, the nucleotide at position 1 of the 5' end of the antisense strand of the dsRNA is a modified nucleotide, where the modified nucleotide is a chemically modified nucleotide represented by formula (II). [ka] Here, B represents the base at the position corresponding to the nucleotide at position 1 of the 5' end of the antisense strand. In some specific embodiments, B represents uracil.

[0036] In some embodiments, the antisense strand is at least partially reverse-complementary to the target sequence in order to mediate RNA interference. In some embodiments, there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer mismatches between the antisense strand and the target sequence, and in some embodiments, the antisense strand is completely reverse-complementary to the target sequence.

[0037] In some embodiments, the sense strand and the antisense strand are at least partially inversely complementary to form a double-stranded region; in some embodiments, there are 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, and 1 or fewer mismatches between the sense strand and the antisense strand; and in some embodiments, the sense strand is completely inversely complementary to the antisense strand.

[0038] In some embodiments, the sense strand and the antisense strand each independently have 16-35, 16-34, 17-34, 17-33, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 19-25, 19-24, or 19-23 nucleotides.

[0039] In some specific embodiments, the sense strand and antisense strand each have independently 19, 20, 21, 22, or 23 nucleotides.

[0040] In some embodiments, the lengths of the sense strand and the antisense strand are the same or different, with the sense strand having 19 to 23 nucleotides and the antisense strand having 19 to 26 nucleotides. In some embodiments, the sense chain to antisense chain length ratio may be 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the length ratio of the sense chain to the antisense chain is 19 / 21, 21 / 23, or 23 / 25.

[0041] In some embodiments, the dsRNA includes one or two blunt ends.

[0042] In some embodiments, the dsRNA contains 1 to 4 unpaired nucleotides, for example, 1, 2, 3, or 4 overhanging ends.

[0043] In some embodiments, the dsRNA includes a protruding end at the 3' end of the antisense strand.

[0044] In some embodiments, the sense strand of the above dsRNA is 5'-N a N a N a N a N a N a N b N b N b N a N a N a N a N a N a N a Na N a N a comprises, or is, a nucleotide sequence represented by the formula -3’, where N a is a nucleotide modified with a 2’-methoxy group, and N b is a nucleotide modified with 2’-fluoro.

[0045] In some embodiments, the antisense strand of the dsRNA is 5’-N a ’N b ’N a ’X’N a ’X’N a ’N a ’N a ’X’N a ’X’N a ’N b ’N a ’X’N a ’X’N a ’N a ’N a ’-3’ comprises, or is, a nucleotide sequence represented by the formula, where each X’ is independently N a ’ or N b ’, and N a ’ is a nucleotide modified with a 2’-methoxy group, and N b ’ is a nucleotide modified with 2’-fluoro.

[0046] In some embodiments, the antisense strand of the dsRNA is 5’-N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’Nb ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’N b ’Na 'N a 'N a 'N a 'N a 'N a 'N a '-3', 5'-N a 'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a '-3', 5'-N a 'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a '-3' It contains the nucleotide sequence shown in the formula, or is the above nucleotide sequence, Here, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' is a nucleotide modified with 2'-fluoro.

[0047] In some embodiments, the antisense strand of the above dsRNA is 5'-Na 'N b 'N a 'X'N a 'X'W'N a 'N a 'X'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a It contains the nucleotide sequence represented by the formula '-3', or the above nucleotide sequence, where each X' is independently N a 'or N b ' and N a ' is a nucleotide modified with a 2'-methoxy group, N b ' represents a nucleotide modified with 2'-fluoro, and W' represents a nucleotide containing the chemical modification shown in formula (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof.

[0048] In some embodiments, the antisense strand of the above dsRNA is 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a '-3', 5'-N a 'N b 'N a 'N b 'N a 'N b 'W'N a 'N a 'N b 'Na ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N b ’N a ’N b ’W’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N a ’N a ’N a ’Na ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N b ’N a ’N b ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N b ’N a ’N a ’N a ’N b ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N a ’N a ’N b ’N a ’N b ’Na ’N b ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N a ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N b ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N a ’N a ’N a ’N a ’N a ’N a ’-3’、 5’-N a ’N b ’N a ’N a ’N a ’N a ’W’N a ’N a ’N a ’N a ’N a ’N a ’N b ’N a ’N b ’Na 'N a 'N a 'N a 'N a '-3' It contains the nucleotide sequence shown in the formula, or is the above nucleotide sequence, Here, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' represents a nucleotide modified with 2'-fluoro, and W' represents a nucleotide containing the chemical modification shown in formula (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, the antisense strand of the above dsRNA is 5'-V'N b 'N a 'X'N a 'X'N a 'N a 'N a 'X'N a 'X'N a 'N b 'N a 'X'N a 'X'N a 'N a 'N a It contains the nucleotide sequence represented by the formula '-3', or the above nucleotide sequence, where each X' is independently N a 'or N b ' and N a ' is a nucleotide modified with a 2'-methoxy group, N b ' represents a nucleotide modified with 2'-fluoro, and V' represents a chemically modified nucleotide shown in formula (II).

[0050] In some embodiments, the antisense strand of the above dsRNA is 5'-V'N b 'N a 'N a 'N a 'N b 'N a 'N a 'N a 'N a 'Na 'N b 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a '-3', 5'-V'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N a 'N b 'N a 'N b 'N a 'N a 'N a 'N a 'N a 'N a 'N a '-3' It contains the nucleotide sequence shown in the formula, or is the above nucleotide sequence, Here, N a ' is a nucleotide modified with a 2'-methoxy group, N b ' represents a nucleotide modified with 2'-fluoro, and V' represents a chemically modified nucleotide shown in formula (II).

[0051] In some embodiments, the chemical modifications shown in formulas (I), (I-1), (I-2) or their pharmaceutically acceptable salts are: [ka] Selected from, where B represents the base at position 7 of the 5' end of the antisense strand, and in some specific embodiments, B is adenine.

[0052] In some embodiments, the chemical modifications shown in formulas (I'), (I'-1), (I'-2) or their pharmaceutically acceptable salts are: [Chemical formula] selected from, where M is O or S, where B represents the base at the 7th position of the 5'-end of the antisense strand, and in some specific embodiments, B is adenine.

[0053] In some embodiments, M is S. In some specific embodiments, M is O.

[0054] In some embodiments, the chemically modified nucleotide shown in formula (II) is [Chemical formula] selected from, where B represents the base at the position corresponding to the nucleotide at the 1st position of the 5'-end of the antisense strand. In some specific embodiments, B represents uracil.

[0055] In some embodiments, the chemically modified nucleotide shown in formula (II) is [Chemical formula] selected from, where B represents the base at the position corresponding to the nucleotide at the 1st position of the 5'-end of the antisense strand. In some specific embodiments, B represents uracil.

[0056] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand of the above dsRNA is a phosphodiester group having a modifying group.

[0057] In some embodiments, at least one phosphodiester group in the sense strand and / or antisense strand of the above dsRNA is a thiophosphodiester group.

[0058] In some embodiments, the above thiophosphodiester group is Between the first and second nucleotides at the 5' end of the sense strand mentioned above, Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 3' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, and It is located at at least one of the positions between the second and third nucleotides at the 3' end of the antisense strand mentioned above.

[0059] In some embodiments, the sense chain and / or antisense chain contains a plurality of thiophosphodiester groups, and the thiophosphodiester groups are Between the first and second nucleotides at the 5' end of the sense strand, and Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 3' end of the sense strand, and Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, It is located between the second and third nucleotides at the 3' end of the antisense strand mentioned above.

[0060] In some embodiments, the sense chain and / or antisense chain contains a plurality of thiophosphodiester groups, and the thiophosphodiester groups are Between the first and second nucleotides at the 5' end of the sense strand, and Between the second and third nucleotides at the 5' end of the sense strand mentioned above, Between the first and second nucleotides at the 5' end of the antisense strand mentioned above, Between the second and third nucleotides at the 5' end of the antisense strand mentioned above, Between the first and second nucleotides at the 3' end of the antisense strand mentioned above, It is located between the second and third nucleotides at the 3' end of the antisense strand mentioned above.

[0061] In some embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from any one of the nucleotide sequences shown in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NOs. 22 to 33, and SEQ ID NO: 35.

[0062] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 16.

[0063] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 20.

[0064] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 22.

[0065] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 23.

[0066] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 24.

[0067] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 25.

[0068] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 26.

[0069] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 27.

[0070] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 28.

[0071] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 29.

[0072] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 30.

[0073] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 31.

[0074] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 32.

[0075] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 33.

[0076] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 6, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 35.

[0077] In some embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from any one of SEQ ID NOs: 9 to 20 or SEQ ID NOs: 33 to 35.

[0078] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 9.

[0079] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 10.

[0080] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 11.

[0081] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 12.

[0082] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 13.

[0083] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 14.

[0084] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 15.

[0085] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 16.

[0086] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 17.

[0087] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 18.

[0088] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 19.

[0089] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 20.

[0090] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 33.

[0091] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 34.

[0092] In some specific embodiments, the dsRNA has a sense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 7, and an antisense strand that includes or is selected from the nucleotide sequence shown in SEQ ID NO: 35.

[0093] In some embodiments, the above dsRNA is The sense strand's nucleotide sequence includes SEQ ID NO: 6, and the antisense strand's nucleotide sequence includes SEQ ID NO: 16.

[0094] In some embodiments, the dsRNA has a sense strand nucleotide sequence shown in SEQ ID NO: 6 and an antisense strand nucleotide sequence shown in SEQ ID NO: 16. The disclosure further provides a dsRNA complex comprising any one of the dsRNAs and a target ligand linked to the dsRNA.

[0095] In some embodiments, the dsRNA covalently or noncovalently binds to the target ligand.

[0096] In some embodiments, the target ligand targets the liver. In some embodiments, the target ligand binds to the asialoclycoprotein receptor (ASGPR). In some embodiments, the target ligand comprises a galactose cluster or a galactose derivative cluster, the galactose derivative being selected from N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0097] In some embodiments, the target ligand is ligated to the 3' end of the sense strand of the dsRNA.

[0098] In some embodiments, the target ligand is ligated to the dsRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group.

[0099] In some embodiments, the target ligand is indirectly ligated to the dsRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group.

[0100] In some embodiments, the target ligand is directly ligated to the dsRNA terminus via a phosphodiester group, a thiophosphodiester group, or a phosphonic acid group.

[0101] In some embodiments, the target ligand is directly ligated to the end of the dsRNA sense strand via a phosphodiester group or a thiophosphodiester group.

[0102] In some embodiments, the target ligand is directly ligated to the 3' end of the dsRNA sense strand via a phosphodiester group or a thiophosphodiester group.

[0103] In some embodiments, a lipophilic group such as cholesterol can be introduced to the end of the sense strand to promote the entry of dsRNA into cells. This lipophilic group can covalently bind to small interfering nucleic acids. For example, introducing cholesterol, lipoprotein, or vitamin E to the end contributes to interaction with intracellular mRNA via the cell membrane, which is composed of a lipid bilayer. Furthermore, dsRNA may be modified with non-covalent bonds, for example, by binding to phospholipid molecules, polypeptides, cationic polymers, etc., via hydrophobic or ionic bonds, thereby improving its stability and biological activity.

[0104] In some embodiments, the target moiety of a target ligand consists of one or more target groups or target moieties, and the target ligand cooperates to guide the therapeutic reagent to which it is ligated to be delivered to a desired target site. In some cases, the target moiety can bind to cells or cell receptors and also initiate endocytosis to facilitate the entry of the therapeutic reagent into the cell. The target moiety may include a compound that has affinity for cell receptors or cell surface molecules or antibodies. Various target ligands containing target moieties can be ligated to therapeutic reagents and other compounds so that the reagent targets cells and specific cell receptors.

[0105] In some embodiments, the target moiety type includes carbohydrates, cholesterol, and cholesteryl groups or steroids. Target moieties capable of binding to cell receptors include sugars, such as galactose, galactose derivatives (e.g., N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, N-isobutyrylgalactosamine), mannose, and mannose derivatives).

[0106] The target moiety that binds to the asialocrypoprotein receptor (ASGPR) is known to be particularly useful for inducing the delivery of oligomeric compounds to the liver. The asialocrypoprotein receptor is expressed in large quantities in liver cells (hepatocytes). The target moiety of ASGPR-targeting cell receptors includes galactose and galactose derivatives. Specifically, galactose derivative clusters include clusters consisting of 2, 3, 4, or 4 or more N-acetyl-galactosamine (GalNAc or NAG) molecules, which can promote the uptake of certain compounds in hepatocytes. The GalNAc cluster coupled to the oligomeric compound is intended to guide the composition to the liver, where the N-acetyl-galactosamine sugar can bind to the asialocrypoprotein receptor on the surface of liver cells. Binding to the asialocrypoprotein receptor is thought to activate receptor-mediated endocytosis, thereby promoting the entry of the compound into the cell.

[0107] In some embodiments, the target ligand may contain two, three, four, or more target moieties.

[0108] In some embodiments, each target moiety independently comprises a galactosamine derivative, which is N-acetyl-galactosamine. Other sugars available as target moieties and having affinity for the asialoglycoprotein receptor may be selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, Nn-butyryl-galactosamine, and N-isobutyryl-galactosamine.

[0109] In some embodiments, the target ligand in this disclosure comprises N-acetylgalactosamine as the target moiety. [ka]

[0110] In some embodiments, the target ligand comprises three terminal galactosamines or galactosamine derivatives (e.g., N-acetyl-galactosamines) each having affinity for an asial glycoprotein receptor. In some embodiments, the target ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as the target moiety.

[0111] In some embodiments, the target ligand comprises four terminal galactosamines or galactosamine derivatives (e.g., N-acetyl-galactosamines) each having affinity for an asialoclycoprotein receptor. In some embodiments, the target ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as the target moiety.

[0112] When referring to three terminal N-acetylgalactosamines, commonly used terms in this field include tri-antennary, tri-valent, and trimer.

[0113] When referring to four-terminal N-acetylgalactosamines, commonly used terms in this field include tri-antennary, tri-valent, and tetramer.

[0114] In some embodiments, the target ligand provided herein is a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof. [ka]

[0115] In some embodiments, the target ligand provided herein is a compound represented by formula (III-2) or a pharmaceutically acceptable salt thereof. [ka]

[0116] In some embodiments, the N-acetyl-galactosamine moiety of the target ligand can be substituted with N-trifluoroacetylgalactosamine, N-propionylgalactosamine, Nn-butyrylgalactosamine, or N-isobutyrylgalactosamine.

[0117] In some embodiments, the dsRNA complex has a sense strand nucleotide sequence containing or selected from the above nucleotide sequence, and an antisense strand nucleotide sequence containing or selected from any one of the following nucleotide sequences: SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NOs. 22 to 33, and SEQ ID NO: 35.

[0118] In some embodiments, the dsRNA complex has a sense strand nucleotide sequence that includes or is selected from the above nucleotide sequences, and an antisense strand nucleotide sequence that includes or is selected from any one of the nucleotide sequences of SEQ ID NOs: 9 to 20 or SEQ ID NOs: 33 to 35.

[0119] In some embodiments, the dsRNA complex has a sense strand nucleotide sequence containing or selected from the above nucleotide sequence, and an antisense strand nucleotide sequence containing or selected from any one of the above nucleotide sequences: SEQ ID NO: 16, SEQ ID NO: 20, or SEQ ID NO: 33.

[0120] In some embodiments, the dsRNA complex is The sense strand's nucleotide sequence includes SEQ ID NO: 3, and the antisense strand's nucleotide sequence includes SEQ ID NO: 16.

[0121] In some embodiments, the dsRNA complex is The nucleotide sequence of the sense strand is shown in Sequence ID No. 3, and the nucleotide sequence of the antisense strand is shown in Sequence ID No. 16.

[0122] In some embodiments, the dsRNA complex has the following structure or a pharmaceutically acceptable salt thereof: [ka] Here, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Gf = guanine 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gm = guanine 2'-OMe ribonucleoside, and Um = uracil 2'-OMe ribonucleoside.

[0123] [ka] represents a thiophosphodiester group, [ka] This represents a phosphodiester group, NAG0052' is, [ka] It represents.

[0124] In some embodiments, the dsRNA complex has the following structure or a pharmaceutically acceptable salt thereof: [ka] Here, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Gf = guanine 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gm = guanine 2'-OMe ribonucleoside, and Um = uracil 2'-OMe ribonucleoside.

[0125] [ka] This represents the thiophosphodiester group anionic form, [ka] This represents the phosphodiester group anion form, NAG0052' is, [ka] It represents.

[0126] In some embodiments, the pharmaceutically acceptable salt may be a conventional salt in the art, and includes, but is not limited to, sodium salts, potassium salts, ammonium salts, amine salts, and the like.

[0127] In some embodiments, the above dsRNA complex is TJR100422, TJR100423, TJR100424, TJR100425, TJR100426, TJR100427, TJR100428, TJR100429, TJR100430, TJR100431, TJR100432, TJR100800, TJR100801, TJR100802, TJR100803, TJR100804, TJR100805 It is one of the following selected from TJR100806, TJR100807, TJR100808, TJR100809, TJR100810, TJR100811, TJR101079, TJR101080, TJR101081, TJR101082, TJR101083, TJR101085, TJR101086, TJR101087, TJR101088, TJR101084, TJR102134, and TJR102136.

[0128] In some embodiments, the above dsRNA complex is TJR101079, and its structure is: [ka] Here, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = uracil 2'-F ribonucleoside, Gf = guanine 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gm = guanine 2'-OMe ribonucleoside, and Um = uracil 2'-OMe ribonucleoside.

[0129] [ka] This represents the thiophosphodiester group anionic form, [ka] This represents the phosphodiester group anion form, NAG0052' is, [ka] It represents.

[0130] Another aspect of the present disclosure provides a composition comprising the dsRNA and / or dsRNA complex and one or more pharmaceutically acceptable excipients, such as carriers, transporters, diluents, and / or delivery polymers.

[0131] In this disclosure, various drug delivery systems are known and applicable to the dsRNA or dsRNA complexes of this disclosure, such as packaging into liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated cell endocytosis, reverse transcription viruses, or the construction of nucleic acids that become part of other vectors.

[0132] Another aspect of this disclosure provides the use of the above-mentioned dsRNA and / or dsRNA complexes or compositions thereof in the preparation of agents for treating a disease of interest, which in some embodiments are selected from liver-derived diseases.

[0133] Another aspect of this disclosure provides a method for treating a disease of interest, comprising administering the dsRNA and / or dsRNA complex or composition to the subject.

[0134] Another aspect of this disclosure provides a method for inhibiting LPA mRNA expression in a subject, comprising administering the dsRNA and / or dsRNA complex or composition to the subject.

[0135] Another aspect of this disclosure provides a method for in vivo delivering an expression inhibitory oligomer compound to the liver, wherein the complex or composition is administered as the target.

[0136] The dsRNAs, dsRNA complexes, compositions, and methods disclosed herein can reduce the level of target mRNA in cells, cell populations, tissues, or subjects by administering a therapeutically effective amount of the dsRNAs described herein to the subject, wherein the dsRNAs are ligated to target ligands, thereby inhibiting the expression of target mRNA in the subject.

[0137] In some embodiments, the subject has already been identified as having abnormal expression of the target gene in the target cells or tissue.

[0138] The subjects described in this disclosure are those suffering from a disease or condition that would benefit from the reduction or inhibition of target mRNA expression.

[0139] Delivery may be by local administration (e.g., direct injection, implantation, or local administration), systemic administration, or by subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, percutaneous, airway (aerosol), nasal, oral, rectal, or local (including oral, cheek, and sublingual) administration.

[0140] In a selective embodiment, the pharmaceutical compositions provided herein may be administered by injection, for example, intravenous, intramuscular, intradermal, subcutaneous, duodenal, or intraperitoneal injection.

[0141] In a selective embodiment, after the target ligand is ligated to the dsRNA to form a complex, the complex may be packaged in a reagent kit.

[0142] In another embodiment, the Disclosure provides a pharmaceutical composition comprising the dsRNA and / or dsRNA complex described herein.

[0143] In some embodiments, the pharmaceutical composition may further contain pharmaceutically acceptable additives and / or adjuvants, which may be one or more of the various formulations or compounds commonly used in the art. For example, the pharmaceutically acceptable additives may include at least one of pH buffers, protective agents, and osmotic regulators.

[0144] In some embodiments, when the dsRNA, dsRNA complexes, or pharmaceutical compositions described herein come into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods, such as immunofluorescence analysis methods including Western Blot or flow cytometry, the dsRNA, dsRNA complexes, or pharmaceutical compositions inhibit the expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0145] In some embodiments, when the dsRNA, dsRNA complex, or pharmaceutical composition thereof described herein comes into contact with cells expressing a target gene, the excess expression percentage of the target gene mRNA induced by the dsRNA, dsRNA complex, or pharmaceutical composition is measured by, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods based on, for example, PCR or branched DNA (bDNA), or protein-based methods, such as immunofluorescence analysis methods such as Western Blot or flow cytometry, and is 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0146] In some embodiments, when the dsRNA, dsRNA complexes, or their pharmaceutical compositions described herein come into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods based on PCR or branched DNA (bDNA), or by protein-based methods such as immunofluorescence analysis including Western Blot or flow cytometry, the dsRNA, dsRNA complexes, or their pharmaceutical compositions retain on-target activity while simultaneously reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0147] In some embodiments, when the dsRNA, dsRNA complexes, or their pharmaceutical compositions described herein come into contact with cells expressing a target gene, for example, by psiCHECK activity screening and luciferase reporter gene detection methods, as well as by methods such as PCR, branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis methods including Western Blot and flow cytometry, the dsRNA, dsRNA complexes, or their pharmaceutical compositions reduce on-target activity by at least 20%, at least 19%, at least 15%, at least 10%, at least 5%, or more than 1%, while simultaneously reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0148] In some embodiments, when the dsRNA and / or dsRNA complexes or their pharmaceutical compositions described herein come into contact with cells expressing a target gene, for example, psiCHECK activity screening and luciferase reporter gene detection methods, as well as methods based on PCR or branched DNA (bDNA), or protein-based methods, such as Western When measured by immunofluorescence analysis methods such as blotting or flow cytometry, dsRNA, dsRNA complexes, or their pharmaceutical compositions enhance on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, while simultaneously reducing off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0149] In a further specific embodiment, when the dsRNA and / or dsRNA complex or the pharmaceutical composition thereof described herein comes into contact with cells expressing a target gene, IC 50 nM is a range of 1nM or less, 0.9nM or less, 0.8nM or less, 0.7nM or less, 0.6nM or less, 0.5nM or less, 0.4nM or less, 0.3nM or less, 0.2nM or less, 0.19nM or less, 0.18nM or less, 0.17nM or less, 0.16nM or less, 0.15nM or less, 0.14nM or less, 0.13nM or less, 0.12nM or less, 0.11nM or less, 0.10nM or less, 0.09nM or less, 0.08nM or less, 0.07nM or less, 0.06nM or less, 0.05nM or less, 0.04nM or less, 0.03nM or less, 0.02nM or less, 0.01nM or less, or any range between any two values.

[0150] This disclosure further provides cells comprising the dsRNA and / or dsRNA complex of this disclosure.

[0151] The Disclosure further provides a kit or reagent kit comprising one or more containers, wherein each container independently contains the dsRNA and / or dsRNA complexes of the Disclosure, or a pharmaceutical composition thereof.

[0152] This disclosure provides a method for silencing a target gene or mRNA of a target gene in a cell, further comprising the step of introducing a dsRNA and / or dsRNA complex and / or pharmaceutical composition according to this disclosure into the cell.

[0153] This disclosure further provides a method for silencing a target gene or mRNA of a target gene in cells in vivo or in vitro, the method comprising the step of introducing a dsRNA and / or dsRNA complex and / or pharmaceutical composition according to this disclosure into the cells.

[0154] The present disclosure further provides a method for inhibiting a target gene or the mRNA expression of a target gene, comprising administering an effective amount or effective dose of the dsRNA and / or dsRNA complex and / or pharmaceutical composition according to the present disclosure to a subject requiring such inhibition.

[0155] In some embodiments, administration is carried out by methods of administration including intramuscular, intrabronchial, intrathoracic, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal fluid, or a combination thereof.

[0156] In some embodiments, the effective amount or effective dose of dsRNA and / or dsRNA complex and / or pharmaceutical composition is approximately 0.001 mg / kg body weight to approximately 200 mg / kg body weight, approximately 0.01 mg / kg body weight to approximately 100 mg / kg body weight, or approximately 0.5 mg / kg body weight to approximately 50 mg / kg body weight.

[0157] In some embodiments, the target gene is LPA.

[0158] In another embodiment, the present disclosure provides the use of the above-mentioned dsRNA and / or dsRNA complex or a pharmaceutical composition comprising the above-mentioned dsRNA and / or dsRNA complex in the preparation of a drug.

[0159] In another embodiment, the Disclosure provides the dsRNA and / or pharmaceutical composition and / or dsRNA complex for treating and / or preventing diseases associated with elevated lipoprotein(a) and / or apolipoprotein(a) levels in a subject. In some embodiments, the diseases associated with elevated lipoprotein(a) and / or apolipoprotein(a) levels are selected from cardiovascular diseases. In some embodiments, the cardiovascular diseases are selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0160] In another embodiment, the present disclosure provides the dsRNA and / or pharmaceutical composition and / or dsRNA complex for treating and / or preventing a disease, wherein the disease is selected from cardiovascular diseases. In some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0161] This disclosure provides the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex for reducing lipoprotein(a) and / or apolipoprotein(a) levels.

[0162] This disclosure provides the use of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex in the preparation of a drug for inhibiting LPA expression.

[0163] This disclosure provides the use of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex in the preparation of agents for treating and / or preventing diseases associated with elevated lipoprotein(a) and / or apolipoprotein(a) levels in a subject. In some embodiments, the diseases associated with elevated lipoprotein(a) and / or apolipoprotein(a) levels are selected from cardiovascular diseases. In some embodiments, the cardiovascular diseases are selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0164] This disclosure provides the use of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex in the preparation of agents for treating and / or preventing a disease, wherein the disease is selected from cardiovascular diseases. In some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0165] This disclosure provides the use of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex in the preparation of agents for reducing lipoprotein(a) and / or apolipoprotein(a) levels.

[0166] This disclosure provides a method for inhibiting LPA expression, comprising administering an effective amount or effective dose of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex to the target.

[0167] This disclosure provides a method for treating and / or preventing a disease associated with elevated lipoprotein(a) and / or apolipoprotein(a) levels in a subject, comprising administering an effective amount or effective dose of the dsRNA and / or pharmaceutical composition and / or dsRNA complex to the subject. In some embodiments, the disease associated with elevated lipoprotein(a) and / or apolipoprotein(a) levels is selected from cardiovascular diseases, and in some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0168] This disclosure provides a method for treating and / or preventing a disease, comprising administering an effective amount or effective dose of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex to a target, wherein the disease is selected from cardiovascular diseases. In some embodiments, the cardiovascular disease is selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease or aortic stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure.

[0169] This disclosure provides a method for reducing lipoprotein(a) and / or apolipoprotein(a) levels, comprising administering an effective amount or effective dose of the above-mentioned dsRNA and / or pharmaceutical composition and / or dsRNA complex to the target.

[0170] This disclosure provides a method for in vivo delivery of a dsRNA that inhibits LPA expression and / or replication to the liver, wherein the dsRNA and / or pharmaceutical composition and / or dsRNA complex are administered as the target.

[0171] In another embodiment, the Disclosure further provides a method for preparing the dsRNA and / or dsRNA complex or pharmaceutical composition described herein, which includes synthesizing the dsRNA and / or dsRNA complex or pharmaceutical composition described herein.

[0172] The Disclosure further provides dsRNAs or dsRNA complexes characterized by substituting base T for one or more bases U in any one of the dsRNAs or dsRNA complexes of the Disclosure, for example, 1, 2, 3, 3, 5, 6, 7, 8, 9, or 10 bases U. In some embodiments, all bases U in the Disclosure may be substituted with base T.

[0173] The medicinal salts of the compounds described herein are selected from inorganic salts or organic salts, and the compounds described herein can react with acidic or basic substances to produce the corresponding salts.

[0174] The compounds of this disclosure may be in the form of specific geometric or stereoisomers. This disclosure includes all cis-trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as mixtures rich in enantiomers or diastereomers, and all such compounds are intended to be within the scope of this disclosure. Substituents such as alkyl groups may have other chiral carbon atoms. All such isomers and mixtures thereof are within the scope of this disclosure. Compounds containing chiral carbon atoms of this disclosure may be isolated in the form of optically active pures or in racemic forms. The optically active pures may be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0175] Optically active (R)- and (S)- isomers and D- and L isomers can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain one enantiomer of a compound in this disclosure, it can be prepared by asymmetric synthesis or by induction with a chiral auxiliary agent, where the resulting diastereomer mixture is isolated and the auxiliary groups are cleaved to provide the pure enantiomer. Alternatively, if the molecule contains a basic functional group (e.g., an amino group) or an acidic functional group (e.g., a carboxyl group), a salt of the diastereomer is formed with a suitable optically active acid or base, and the diastereomer is cleaved by conventional methods well known in the art, and then recovered to obtain the pure enantiomer. The isolation of enantiomers and diastereomers is usually completed by chromatography, which employs a chiral stationary phase and is optionally combined with a chemical induction method (e.g., generating a carbamate from an amine).

[0176] JPEG2026520380000029.jpg35161

[0177] JPEG2026520380000030.jpg17160

[0178] Furthermore, unless otherwise specified, the compounds and intermediates of this disclosure may exist in different tautomer forms, and all such forms are included within the scope of this disclosure. The terms “tautomer” or “tautomer form” refer to structural isomers of different energies that can be interconverted over a low energy barrier.

[0179] This disclosure is the same as described herein, but further includes several isotopically labeled compounds in which one or more atoms are substituted with atoms having atomic weights or mass numbers different from those commonly found in nature. Examples of isotopes that can be bound to the compounds of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, for example, respectively 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Examples include Cl.

[0180] Compared to non-deuterated drugs, deuterated drugs offer advantages such as reduced toxicity and side effects, increased drug stability, improved therapeutic efficacy, and extended biological half-life. All isotopic transformations of the compounds relating to this disclosure, whether radioactive or non-radioactive, are included within the scope of this disclosure. Each available hydrogen atom bonded to a carbon atom may be independently substituted with a deuterium atom, where the deuterium substitution may be partial or complete, with partial deuterium substitution meaning that at least one hydrogen atom is substituted with at least one deuterium atom.

[0181] Unless otherwise stated, if one position of a compound in the present disclosure is specifically designated as “deuterium” or “D”, that position should be understood to have a deuterium abundance at least 1000 times higher than the natural abundance of deuterium (which is 0.015%) (i.e., at least 15% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 1000 times higher than the natural abundance of deuterium (i.e., at least 15% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 2000 times higher than the natural abundance of deuterium (i.e., at least 30% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 3000 times higher than the natural abundance of deuterium (i.e., at least 45% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 3340 times higher than the natural abundance of deuterium (i.e., at least 50.1% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 3500 times higher than the natural abundance of deuterium (i.e., at least 52.5% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 4000 times higher than the natural abundance of deuterium (i.e., at least 60% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 4500 times higher than the natural abundance of deuterium (i.e., at least 67.5% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 5000 times higher than the natural abundance of deuterium (i.e., at least 75% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 5500 times higher than the natural abundance of deuterium (i.e., at least 82.5% deuterium is incorporated).In some embodiments, each designated deuterium atom has a deuterium abundance at least 6000 times higher than the natural abundance of deuterium (i.e., at least 90% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 6333.3 times higher than the natural abundance of deuterium (i.e., at least 95% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 6466.7 times higher than the natural abundance of deuterium (i.e., at least 97% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 6600 times higher than the natural abundance of deuterium (i.e., at least 99% deuterium is incorporated). In some embodiments, each designated deuterium atom has a deuterium abundance at least 6633.3 times higher than the natural abundance of deuterium (i.e., at least 99.5% deuterium is incorporated). Those skilled in the art can synthesize compounds in their deuterated form by referring to relevant literature. When preparing compounds in their deuterated form, commercially available deuterated starting materials may be used, or they may be synthesized using deuterating reagents by conventional techniques, and these deuterating reagents include, but are not limited to, borane deuterated, borane-tetrahydrofuran trihydrogenated solution, lithium aluminum hydride deuterated, iodoethane deuterated, and iodomethane deuterated.

[0182] The terms “approximately” and “nearly” mean that the numerical value is within the acceptable margin of error of a specific value measured by a person skilled in the art, and the numerical portion is determined by how it is measured or measured (i.e., the limits of the measuring system). For example, “approximately” may mean a standard deviation of 1 or greater than 1. Alternatively, “approximately” or “basically includes” may mean that it varies within a range of at most 20%, for example, between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, or between 0.5% and 1%, and in this disclosure, wherever the term “approximately” precedes a number or numerical range, it includes a predetermined number of embodiments. Unless otherwise stated, where specific values ​​appear in this application and claims, the meaning of “approximately” or “basically includes” should be assumed to be within the acceptable margin of error of the specific value.

[0183] This disclosure incorporates the full text of WO2022028462A, WO2023274395A, WO2023208023A, and WO2023109940A.

[0184] Explanation of terms To make this disclosure more easily understood, several technical and scientific terms are defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art.

[0185] Unless otherwise specified, the terms “apolipoprotein(a) gene,” “Apo(a) gene,” “LPA,” and “Lp(a)” are used interchangeably in the context of this disclosure. LPA includes, but is not limited to, human LPA, cynomolgus monkey LPA, mouse LPA, and rat LPA, and its amino acids, complete coding sequences, and mRNA sequences can be readily obtained using previously disclosed databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project site.

[0186] The term "target sequence" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during LPA transcription, including mRNA of the RNA processed product as the main transcript. The targeted portion of the target sequence must be long enough to function as a substrate for iRNA-directed cleavage. In one embodiment, the target sequence is located within the protein-coding region of the LPA.

[0187] As used herein, the sense strand of a dsRNA (also called SS, SS strand, or sense strand) refers to the strand containing a sequence identical or essentially identical to the target mRNA sequence, and the antisense strand of a dsRNA (also called AS or AS strand) refers to the strand having a sequence that is at least partially complementary to the target mRNA sequence.

[0188] In the context of describing the dsRNA sense strands described herein, the term "at least 17 consecutive nucleotides of a sequence that differs from the nucleotide sequence of SEQ ID NO: 1 by three or fewer nucleotides" is intended to indicate that the dsRNA sense strands described herein include at least 17 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1, or a sequence that differs from at least 17 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1 by three or fewer nucleotides, optionally a sequence that differs from two or fewer nucleotides, or optionally a sequence that differs from one nucleotide. Optionally, the dsRNA sense strands described herein include at least 18 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1, or a sequence that differs from at least 18 consecutive nucleotides of the sense strand shown in SEQ ID NO: 1 by three or fewer nucleotides, optionally a sequence that differs from two or fewer nucleotides, or optionally a sequence that differs from one nucleotide.

[0189] In the context of describing the dsRNA antisense strands described herein, the term "at least 19 consecutive nucleotides of a sequence that differs from the antisense strand shown in SEQ ID NO: 2 by three or fewer nucleotides" is intended to indicate that the sequence includes at least 19 consecutive nucleotides of the antisense strand shown in SEQ ID NO: 2 described herein, or sequences that differ from at least 19 consecutive nucleotides of the antisense strand shown in SEQ ID NO: 2 by three or fewer nucleotides, sequences that optionally differ by two or fewer nucleotides, or sequences that optionally differ by one nucleotide.

[0190] In this disclosure, the "5' region," i.e., the "5' end," and the "5' terminus" of the sense strand or antisense strand are used interchangeably. For example, the nucleotides at positions 2-8 of the antisense strand's 5' region may be substituted with the nucleotides at positions 2-8 of the antisense strand's 5' terminus. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense strand or antisense strand are also used interchangeably.

[0191] Unless otherwise specified, in the context of this disclosure, "G", "C", "A", "T", and "U" each represent a nucleotide, and each includes the bases of guanine, cytosine, adenine, thymidine, and uracil, respectively. The lowercase letter m indicates that the nucleotide adjacent to the upstream of the letter m is a nucleotide modified with a methoxy group, the lowercase letter f indicates that the nucleotide adjacent to the upstream of the letter f is a nucleotide modified with a fluoro group, and the lowercase letter s indicates that the two nucleotides adjacent to both sides of the letter s are linked by thiophosphodiester groups.

[0192] As used in this disclosure, the term “2'-fluoro(2'-F) modified nucleotide” refers to a nucleotide formed by substituting a fluoro group for the hydroxyl group at the 2' position of the ribosyl group of the nucleotide, and “non-fluoro modified nucleotide” refers to a nucleotide or nucleotide analog formed by substituting a non-fluoro group for the hydroxyl group at the 2' position of the ribosyl group of the nucleotide.

[0193] As used in this disclosure, the term “nucleotide modified with a 2'-methoxy group (2'-OMe)” refers to a nucleotide formed by substituting a methoxy group for the 2'-hydroxy group of a ribosyl group.

[0194] In the context of this disclosure, a “nucleotide difference” between one nucleotide sequence and another means that the type of base of the nucleotide at the same position in the former is different from that of the latter. For example, if one nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then there is considered to be a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, when a nucleotide at the original position is replaced with a baseless nucleotide or its equivalent, a nucleotide difference is considered to occur at that position.

[0195] As used herein, the terms “complementary” and “reverse complementary” are interchangeable and have meanings known to those skilled in the art, where, in a double-stranded nucleic acid molecule, the bases of one strand pair complementaryly with the bases of the other strand. In DNA, the purine base adenine always pairs with the pyrimidine base thymine (or uracil in RNA), and the purine base guanine always pairs with the pyrimidine base cytosine. Each base pair contains one purine and one pyrimidine. When adenine in one strand always pairs with thymine (or uracil) in the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the strands can be inferred from the sequence of the complementary strands. Accordingly, “mismatch” in this art means that, in a double-stranded nucleic acid, the bases at corresponding positions do not exist paired in a complementary form.

[0196] The term "dsRNA" refers to a double-stranded RNA molecule that includes both a sense strand and an antisense strand capable of RNA interference.

[0197] The terms "chemical modification" or "modification" encompass all chemical changes to nucleotides, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety with another.

[0198] The term "base" includes any known DNA and RNA bases and base analogs, such as purines and pyrimidines, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and their natural analogs.

[0199] The terms "optionally" or "optionally" mean that the event or environment described thereafter may or may not occur, and the description includes both cases in which the event or environment occurs and cases in which it does not. For example, "C where C is optionally substituted with a halogen or cyano group." 1-6 The term "alkyl group" means that a halogen or cyano group may be present, but is not necessarily required, and this description includes cases where the alkyl group is substituted with a halogen or cyano group, and cases where the alkyl group is not substituted with a halogen or cyano group.

[0200] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a linear or branched group containing 1 to 20 carbon atoms, and in some embodiments, is selected from alkyl groups containing 1 to 12 carbon atoms. Non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl This includes n-octyl groups, 2,3-dimethylpentyl groups, 2,4-dimethylpentyl groups, 2,2-dimethylpentyl groups, 3,3-dimethylpentyl groups, 2-ethylpentyl groups, 3-ethylpentyl groups, n-octyl groups, 2,3-dimethylhexyl groups, 2,4-dimethylhexyl groups, 2,5-dimethylhexyl groups, 2,2-dimethylhexyl groups, 3,3-dimethylhexyl groups, 4,4-dimethylhexyl groups, 2-ethylhexyl groups, 3-ethylhexyl groups, 4-ethylhexyl groups, 2-methyl-2-ethylpentyl groups, 2-methyl-3-ethylpentyl groups, n-nonyl groups, 2-methyl-2-ethylhexyl groups, 2-methyl-3-ethylhexyl groups, 2,2-diethylpentyl groups, n-decyl groups, 3,3-diethylhexyl groups, 2,2-diethylhexyl groups, and various branched isomers thereof.In some embodiments, alkyl groups containing 1 to 6 carbon atoms are selected, and non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and the like. The alkyl group may or may not be substituted, and if substituted, the substituent may be substituted at any available linking point, and in some embodiments, the substituent is independently selected from one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, oxo groups, carboxyl groups, or carboxylic acid ester groups.

[0201] The term "alkoxy group" refers to an -O-(alkyl group), where the definition of an alkyl group is as described above. Non-exclusive examples of alkoxy groups include methoxy, ethoxy, propoxy, and butoxy groups. Alkoxy groups may be optionally substituted or unsubstituted. If substituted, the substituents are preferably halogens, hydroxyl groups, oxo, cyano, amino, or C 1-6 Alkyl alkyl group, C 1-6The group is one or more groups independently selected from an alkoxy group, a 3- to 7-membered cycloalkyl group, or a 3- to 7-membered heterocycloalkyl group, and the alkyl group, alkoxy group, cycloalkyl group, or heterocycloalkyl group is optionally substituted with a halogen, hydroxyl group, nitro group, cyano group, or amino group.

[0202] The term "alkylthio group" refers to -S-(alkyl group), where the definition of alkyl group is as described above. Non-limiting examples of alkylthio groups include methylthio group, ethylthio group, propylthio group, and butylthio group. Alkylthio groups may be optionally substituted or unsubstituted, and if substituted, the substituents are preferably independently C 1-6 Alkoxy group, 3-6 membered cycloalkyl group, 3-6 membered heterocycloalkyl group, 3-6 membered cycloalkoxy group, 3-6 membered heterocycloalkoxy group, C 1-6 The group is one or more groups selected from alkylthio groups, 3-6 membered cycloalkylthio groups, and 3-6 membered heterocycloalkylthio groups, and the above alkoxy group, cycloalkyl group, heterocycloalkyl group, cycloalkoxy group, heteroepoxy group, alkylthio group, cycloalkylthio group, and heterocycloalkylthio group may be optionally substituted with a halogen, hydroxyl group, cyano group, or amino group.

[0203] The term "alkenyl group" refers to a linear or branched non-aromatic hydrocarbon group having at least one carbon-carbon double bond and having 2 to 10 carbon atoms. Such a group may have up to 5 carbon-carbon double bonds. For example, a "C2-C6" alkenyl group is defined as an alkenyl group having 2 to 6 carbon atoms. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, and cyclohexenyl groups. The linear, branched, or cyclic portion of an alkenyl group may contain double bonds and may be optionally substituted with 1-, 2-, 3-, 4-, or 5- at any position permitted by normal valence.

[0204] The term "alkynyl group" refers to a linear or branched hydrocarbon group containing 2 to 10 carbon atoms and at least one carbon-carbon triple bond. Up to 5 carbon-carbon triple bonds may be present. Therefore, "C2-C6 alkynyl group" refers to an alkynyl group having 2 to 6 carbon atoms. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl groups. The linear and branched portions of the alkynyl group may contain triple bonds permissible by normal valence and may be optionally substituted with 1-, 2-, 3-, 4-, or 5- at any position permissible by normal valence.

[0205] The terms "cycloalkyl group" or "carbocyclic group" refer to saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituents, where cycloalkyl groups contain 3 to 20 carbon atoms, and in some embodiments, are selected from those containing 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, and cyclohexadienyl groups, while polycyclic cycloalkyl groups include cycloalkyl groups of spirocycles, fused rings, and crosslinked rings. Cycloalkyl groups may be substituted or unsubstituted, and if substituted, the substituents may be substituted at any available linking site, and in some embodiments, independently, halogens, deuterium, hydroxyl groups, oxo, nitro, cyano, and C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 One or more groups selected from a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group, and the above C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0206] The above cycloalkyl ring may be condensed with an aryl group or a heteroaryl group, where the ring linked to the parent structure is a cycloalkyl group, and non-limiting examples include an indanyl group, a tetrahydronaphthyl group, a benzocycloheptanyl group, etc. The cycloalkyl group may be optionally substituted or not substituted, and if substituted, the substituent may, in some embodiments, be independently a halogen, deuterium, a hydroxyl group, an oxo group, a nitro group, a cyano group, or C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 One or more groups selected from a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group, and the above C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0207] The terms "heterocycloalkyl group," "heterocyclic group," or "heterocyclyl group" refer to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituents containing 3 to 20 ring atoms, where one or more ring atoms are nitrogen, oxygen, or S(O). mA heteroatom selected from (where m is an integer from 0 to 2), but without the -OO-, -OS-, or -SS- ring portion, and the remaining ring atoms are carbon. In some embodiments, selected from those containing 3 to 12 ring atoms, where 1 to 4 are heteroatoms, and in some embodiments, selected from those containing 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl groups include pyrrolidinyl group, imidazolidinyl group, tetrahydrofuran group, tetrahydrothienyl group, dihydroimidazolyl group, dihydrofuranyl group, dihydropyrazolyl group, dihydropyrrolyl group, piperidinyl group, piperazinyl group, morpholinyl group, thiomorpholinyl group, homopiperazinyl group, etc. Polycyclic heterocycloalkyl groups include heterocycloalkyl groups of spiro rings, fused rings, and crosslinked rings. Non-limiting examples of "heterocycloalkyl groups" are: [ka] This includes, among others.

[0208] The above heterocycloalkyl ring may be condensed with an aryl group or a heteroaryl group, where the ring linked to the parent structure is a heterocycloalkyl group, and non-limiting examples include: [ka] This includes, among others.

[0209] The heterocycloalkyl group may be optionally substituted or left unsubstituted. If substituted, the substituents may, in some embodiments, be a halogen, deuterium, hydroxyl group, oxo, nitro group, cyano group, or C13. 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 One or more groups selected from a cycloalkenyloxy group, a 5- to 6-membered aryl group, or a heteroaryl group, and the above C 1-6Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-membered to 6-membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5- to 6-membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0210] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or condensed polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, selected from 6- to 12 members in some embodiments, such as the phenyl group and the naphthyl group. The above aryl ring may be condensed with a heteroaryl group, a heterocycloalkyl group, or a cycloalkyl group, where the ring linked to the parent structure is an aryl ring, and non-limiting examples include: [ka] Includes.

[0211] The aryl group may or may not be substituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, carboxyl groups, or carboxylic acid ester groups, and is preferably a phenyl group.

[0212] The term "fused ring aryl group" may refer to an aromatic, unsaturated fused ring structure containing 8 to 14 ring atoms, formed by the sharing of two or more adjacent atoms between two or more cyclic structures, preferably containing 8 to 12 ring atoms. Examples include fully unsaturated fused ring aryl groups such as naphthalene and phenanthrene, and further include partially saturated fused ring aryl groups such as benzo-3 to 8-membered saturated monocyclic cycloalkyl groups and benzo-3 to 8-membered partially saturated monocyclic cycloalkyl groups. "Fused aromatic ring" refers to the ring system within the fused ring aryl group. Specific examples of fused ring aryl groups include, for example, 2,3-dihydro-1H-indenyl, IH-indenyl, 1,2,3,4-tetrahydronaphthyl, and 1,4-dihydronaphthyl.

[0213] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12 members, such as an imidazolyl group, furanyl group, thienyl group, thiazolyl group, pyrazolyl group, oxazolyl group, pyrrolyl group, tetrazolyl group, pyridyl group, pyrimidine group, thiadiazolyl group, pyrazinyl group, etc., preferably an imidazolyl group, pyrazolyl group, pyrimidine group, or thiazolyl group, and more preferably a pyrazolyl group or thiazolyl group. The heteroaryl ring may be condensed with an aryl group, a heterocyclyl group, or a cycloalkyl ring, where the ring linked to the parent structure is a heteroaryl ring. "Heteroaromatic ring" refers to the ring system within the heteroaryl group. Non-limiting examples of heteroaryl groups include: [ka] Includes.

[0214] The heteroaryl group may be optionally substituted or unsubstituted. If substituted, the substituent may be a halogen, deuterium, hydroxyl group, oxo, nitro group, cyano group, or C in some embodiments. 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-6 membered heterocycloalkoxy group, C 3-8 One or more groups independently selected from a cycloalkenyloxy group, a 5-6 membered aryl group, or a heteroaryl group, and the above C 1-6 Alkyl alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyloxy group, C 2-6 Alkynyloxy group, C 3-6 Cycloalkoxy group, 3-6 membered heterocycloalkoxy group, C 3-8 The cycloalkenyloxy group, 5-6 membered aryl group, or heteroaryl group may be optionally substituted with one or more groups selected from halogens, deuterium, hydroxyl groups, oxo groups, nitro groups, or cyano groups.

[0215] The term "alkylamino group" refers to a group having the structure -NH(C1-C12 alkyl group).

[0216] The term "hydroxyalkyl group" refers to an alkyl group that is substituted with one or more hydroxyl groups, where the alkyl group is as defined above.

[0217] The term "hydroxyl group" refers to the -OH group.

[0218] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0219] The term "haloalkyl group" refers to an alkyl group substituted with a halogen, where the alkyl group is as defined above.

[0220] The term "haloalkoxy group" refers to an alkoxy group substituted with a halogen, where the alkoxy group is defined as described above.

[0221] The term "cyano group" refers to -CN.

[0222] The term "nitro group" refers to -NO2.

[0223] The term "oxo" refers to an =O group, for example, where a carbon atom and an oxygen atom are linked by a double bond, forming a ketone or aldehyde group.

[0224] The term "amino group" refers to -NH2.

[0225] The term "carboxyl group" refers to -C(O)OH.

[0226] The terms "flat end" and "blunt end" are interchangeable and refer to the absence of unpaired nucleotides or nucleotide analogs at a given end of a dsRNA, i.e., no nucleotide overhangs. dsRNAs with two blunt ends are often double-stranded throughout their entire length.

[0227] In the context of this disclosure, each modifying group, or a phosphodiester group having a modification, may be substituted with any group that can be linked to an adjacent nucleotide, for example, chemical modification [ka] in [ka] The portion may be replaced with any group that can be linked to an adjacent nucleotide.

[0228] The term "covalent linkage" refers to a connection between two molecules, whether by covalent bonds or non-covalent bonds (e.g., hydrogen bonds or ionic bonds), and includes both direct and indirect linkages.

[0229] The term "direct linkage" refers to a linkage between a first compound or group and a second compound or group without the involvement of any atoms or groups of atoms.

[0230] The term "indirect linking" refers to the linking of a first compound or group and a second compound or group via an intermediate group, compound, or molecule (e.g., a linking group).

[0231] The term "substituted" indicates that one or more hydrogen atoms on a particular atom (usually carbon, oxygen, and nitrogen atoms) are substituted with any group as defined herein, provided that the substitution does not exceed the normal valence of the particular atom and that the substitution forms a stable compound. Non-exclusive examples of substituents include C1-C6 alkyl groups, C2-C6 alkenyl groups, C2-C6 alkynyl groups, cyano groups, hydroxyl groups, oxo groups, carboxyl groups, cycloalkyl groups, cycloalkenyl groups, heterocyclyl groups, heteroaryl groups, aryl groups, ketones, alkoxycarbonyl groups, aryloxycarbonyl groups, heteroaryloxycarbonyl groups, or halogens (e.g., F, Cl, Br, I). When the substituent is a ketone or oxo (i.e., =O), two (2) hydrogen atoms on the atom are substituted.

[0232] "Substituted with one or more substituents" means that the substituent may be replaced with one or more substituents. If it is replaced with multiple substituents, they may be multiple identical substituents, or a combination of one or more different substituents.

[0233] "Pharmaceutical composition" indicates a mixture of one or more compounds described herein or their physiologically medicinal salts or prodrugs with other chemical components, and other components such as physiologically medicinal carriers and excipients. The pharmaceutical composition is intended to facilitate administration to a living organism, contribute to the absorption of the active ingredient, and further exert biological activity.

[0234] "Pharmacologically acceptable excipients" include, but are not limited to, any approved excipients, carriers, flow enhancers, sweeteners, diluents, preservatives, dyes / colorants, flavorings, surfactants, humectants, dispersants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are permitted for use in humans or livestock.

[0235] As used herein, the term “inhibition” is interchangeable with “reduction,” “silencing,” “downregulate,” “suppression,” and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. The control level may be any type of control level used in this art, for example, a pre-administration baseline level, or a similar level determined from an untreated or control-treated subject, cell, or sample (e.g., buffer control or inactivator control only). For example, the excess mRNA expression level can indicate the degree of inhibition of target gene expression by dsRNA. For instance, excess mRNA expression levels are 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. The inhibition rate of target gene expression can be detected using the Dual-Glo® Luciferase Assay System. The chemiluminescence values ​​of fireflies and sea urchins (Renilla) are read, and the relative value Ratio = Ren / Fir is calculated, with the inhibition rate (%) = 1 - (Ratio + siRNA / Ratioreporter only) × 100%. In this disclosure, the ratio of excess mRNA expression (or excess activity) = 100% - inhibition rate (%).

[0236] Unless otherwise specified, the “compound,” “ligand,” “nucleic acid ligand complex,” “dsRNA complex,” “nucleic acid,” “complex,” “chemical modification,” “target ligand,” “dsRNA,” and “RNAi” of this disclosure may exist independently in the form of a salt, a mixed salt, or an unsalted form (e.g., a free acid or a free base). If existing in the form of a salt or mixed salt, it may be a pharmaceutically acceptable salt.

[0237] The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0238] A "pharmaceutically acceptable acid addition salt" refers to a salt formed with an inorganic or organic acid that can preserve the bioavailability of the free base without other side effects. Inorganic salts include, but are not limited to, hydrochloride, hydrobromide, sulfate, nitrate, and phosphate salts. Organic salts include, but are not limited to, formate, acetate, 2,2-dichloroacetate, trifluoroacetate, propionate, caproate, octanoate, decanoate, undecylenate, glycolate, gluconate, lactate, sebacinate, adipine, glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate, cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, alginate, ascorbate, salicylate, 4-aminosalicylate, and naphthalenedisulfonate salts. These salts can be prepared by methods known in the art.

[0239] A "pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic or organic base that can maintain the bioavailability of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. Salts derived from organic bases include, but are not limited to, the following salts: primary amines, secondary and tertiary amines, substituted amines including naturally substituted amines, cyclic amines, and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, glycine betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamide resins. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared by methods known in the art.

[0240] An "effective dose" or "effective dosage" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also refers to an amount sufficient to enable or facilitate a diagnosis. The effective dose used for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective dose may also be the maximum dose or administration regimen that avoids significant side effects or toxic effects.

[0241] As used herein, “subject,” “patient,” “subject,” or “individual” are interchangeable and include humans or non-human animals, such as mammals like humans and monkeys.

[0242] In this disclosure, ordinal numbers such as "first," "second," etc., are not intended to limit the order or level, but are used solely to distinguish different features, molecules, steps, compositions, elements, etc.

[0243] The dsRNAs provided in this disclosure can be obtained by conventional preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Here, solid-phase synthesis is already available as a commercially available customized service. Modified nucleotide groups can be introduced into the dsRNAs described in this disclosure using nucleoside monomers having the corresponding modifications, and methods for preparing nucleoside monomers having the corresponding modifications and for introducing modified nucleotide groups into dsRNAs are well known to those skilled in the art.

[0244] Some abbreviations used in this disclosure are defined as follows:

[0245] DMF: Dimethylformamide, DIPEA: N-ethyldiisopropylamine, HBTU: Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate DMAP: 4-dimethylaminopyridine, NMI: N-methylimidazole. [Brief explanation of the drawing]

[0246] [Figure 1A] This is the ratio of serum Lp(a) concentration 28 days after administration of TJR101079 and TRD007790 to pre-administration levels. [Figure 1B] This is the ratio of serum Lp(a) concentration 28 days after administration of TJR101079 and TJR102134 to pre-administration levels. [Figure 1C]This is the ratio of serum Lp(a) concentration 28 days after administration of TJR101079 and TJR102136 to pre-administration levels. [Modes for carrying out the invention]

[0247] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure for which specific conditions are not specified will generally be conducted under normal conditions or conditions recommended by the raw material or product manufacturer. In the case of reagents for which a specific source is not specified, such reagents may be obtained from any molecular biology reagent supplier in a quality / purity suitable for molecular biological use.

[0248] Example 1: Chemical Modification [ka] The preparation method for (-)hmpNA(A) was performed according to WO2022028462A. The absolute configuration of (-)hmpNA(A) was (R)-hmpNA(A).

[0249] Example 2: Preparation of NAG0052, L96, and NAG25 [ka] NAG0052, shown in the above structural formula, was prepared according to the method described in patent application WO2023274395A.

[0250] [ka] L96, as shown in the above structural formula, was prepared according to the method described in patent application WO2014025805A.

[0251] [ka] NAG25, shown in the above structural formula, was prepared and obtained according to the method described in patent application WO2017156012A.

[0252] Example 3: Preparation of deuterated 5'-terminated chemically modified phosphoramidites [ka] Step 1 1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-flu[3,2-f][1,3,5,2,4]trioxadisilacyclooctan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-2 [ka] Under a nitrogen gas atmosphere, commercially available compound A1-1 (100 g, 409 mmol) was dissolved in pyridine (1000 mL), and 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSiCl2) (135.6 g, 430.0 mmol) was added at 0°C. The reaction was carried out at 20°C for 18 hours. The reaction mixture was extracted by adding ethyl acetate (1500 mL) and water (1000 mL). The organic phase was washed three times with saturated brine (1000 mL x 3), dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain compound A1-2 (185 g, 380 mmol, yield 92.8%).

[0253] Step 2 3-((benzyloxy)methyl)-1-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-fluoro[3,2-][1,3,5,2,4]trioxadisilacyclooctan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-3 [ka] Compound A1-2 (185 g, 380.1 mmol) was dissolved in N,N-dimethylformamide (1850 mL), then benzyl chloromethyl ether (BOMCl) (89.29 g, 570.1 mmol) and 1,8-diazabicycloundeca-7-ene (DBU) (115.7 g, 760.2 mmol) were added, and the mixture was reacted at 20°C for 3 hours. The reaction mixture was extracted by adding ethyl acetate (3000 mL) and water (3000 mL), and the organic phase was washed three times with saturated brine (2000 mL x 3). After drying over anhydrous sodium sulfate, the mixture was concentrated and purified by column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound A1-3 (target compound, 172 g, 275.6 mmol, yield 72.5%).

[0254] Step 3 3-((benzyloxy)methyl)-1-((6aR,8R,9R,9aR)-2,2,4,4-tetraisopropyl-9-(methoxy-d3)tetrahydro-6H-fluoro[3,2-][1,3,5,2,4]trioxadisilacyclooctan-8-yl)pyrimidine-2,4(1H,3H)-dione A1-4 [ka] Compound A1-3 (150 g, 247.2 mmol) was dissolved in acetonitrile (300 mL), and CD3I (107.5 g, 741.5 mmol) and silver oxide (114.5 g, 494.3 mmol) were added. The mixture was reacted at 55°C for 24 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product, compound A1-4 (target compound, 125 g, 200.3 mmol, yield 81%).

[0255] Step 4 3-((benzyloxy)methyl)-1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(methoxy-d3)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione A1-5 [ka] Compound A1-4 (125 g, 200 mmol) was dissolved in tetrahydrofuran (1250 mL), then pyridine hydrogen fluoride (158.8 g, 1.60 mol) was added, and the mixture was reacted at 25°C for 18 hours. The reaction mixture was extracted by adding ethyl acetate (1000 mL) and water (1000 mL), and the organic phase was washed once with saturated brine (1000 mL). The mixture was then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (dichloromethane:methanol = 20:1) to obtain compound A1-5 (target compound, 59 g, 154.7 mmol, yield 77%).

[0256] Step 5 (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-6 [ka] Compound A1-5 (59 g, 154.7 mmol) was dissolved in pyridine (590 mL), then tert-butyldimethylchlorosilane (TBSCl) (93.2 g, 618 mmol) was added, and the reaction was carried out at 25°C for 18 hours. Next, benzoyl chloride (32.62 g, 232.0 mmol) was added, and the reaction was carried out at 25°C for 3 hours. The reaction mixture was extracted by adding ethyl acetate (1000 mL) and water (1000 mL), and the organic phase was washed twice with saturated brine (1000 mL x 2). After drying over anhydrous magnesium sulfate, the mixture was concentrated and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to obtain compound A1-6 (target compound, 60.0 g, 100.3 mmol, yield 64.6%).

[0257] Step 6 (2R,3R,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2-hydroxymethyl-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-7 [ka] At 0°C, compound A1-6 (60.0 g, 100.3 mmol) was dissolved in methanol (600 mL), then acetyl chloride (10.21 g, 130.1 mmol) was added, and the mixture was reacted at 20°C for 2 hours. Next, silver carbonate was added, and the mixture was stirred at 20°C for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound A1-7 (46.0 g, 94.7 mmol, yield 94.7%).

[0258] Step 7 (2S,3S,4R,5R)-3-(benzyloxy)-5-(3((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-2-carboxylic acid A1-8 [ka] Compound A1-7 (46.0 g, 94.75 mmol) and 4-carbonyl-tetramethylpiperidinyl oxide (TEMPO) (6.95 g, 44.49 mmol) were dissolved in CH3CN (230 mL) and H2O (230 mL). 5(6)-amino-1-(4-aminophenyl)-1,3,3-rimethylindan (PIDA) (67.14 g, 208.4 mmol) was added, and the mixture was reacted at 25°C for 18 hours. Ethyl acetate (500 mL) and water (300 mL) were added to the reaction mixture for extraction. The organic phase was dried over anhydrous sodium sulfate and then concentrated. The mixture was purified by column chromatography (dichloromethane:methanol = 10:1), concentrated under reduced pressure, and compound A1-8 (target compound, 43.0 g, 86 mmol, 90% yield) was obtained.

[0259] Step 8 (3S,4R,5R)-2-acetoxy-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-9 [ka] Compound A1-8 (5 g, 10.0 mmol) was added to a dry flask, purged with argon gas, and then N,N-dimethylformamide (50 mL) was added. Pyridine (8 mL, 100 mmol) and lead acetate (9.7 mL, 50 mmol) were then added in sequence. The reaction mixture was kept in the dark and stirred at room temperature for 48 hours. The reaction was quenched with water (200 mL) and diluted with ethyl acetate (200 mL). The resulting suspension was filtered through a diatomaceous earth pad. The solid was washed with ethyl acetate. The organic layer was separated and concentrated under vacuum. The crude product was purified by C18 reverse-phase column chromatography to obtain the title product A1-9 (1.5 g, yield: 29%) as an α / β mixture.

[0260] MS(ESI): m / z = 514.2[M+H] + .

[0261] Step 9 (3S,4R,5R)-5-(3-((benzyloxy)methyl)-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-2-((diethoxyphosphoryl)methoxy)-4-(methoxy-d3)tetrahydrofuran-3ylbenzoate A1-10 [ka] Under an argon gas atmosphere, diethyl (hydroxymethyl)phosphonate (2.5 g, 156.7 mmol) and diethyl boron trifluoride ether complex (5.7 mL, 43.8 mmol) were added to a solution of compound A1-9 (1.5 g, 2.9 mmol) in anhydrous dichloromethane (15 mL). The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was purified by C18 reverse-phase column chromatography to obtain the title compound A1-10 (1 g, 55% yield).

[0262] MS(ESI): m / z = 622.3[M+H] + .

[0263] Step 10 (3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-ylbenzoate A1-11 [ka] A solution of compound A1-10 (1 g, 14.6 mmol) in TFA (5 mL) was stirred at 80°C for 30 minutes, and then concentrated under vacuum. Title compound A1-11 (1 g, crude product) was obtained and used directly in the next step.

[0264] MS(ESI): m / z = 502.3[M+H] + .

[0265] Step 11 ((((3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-3-hydroxy-4-(methoxy-d3)tetrahydrofuran-2-yl)oxy)methyl)phosphonate diethyl A1-12 [ka] A solution of compound A1-11 (1 g, 2.0 mmol) in ammonia-methanol solution (7 N, 10 mL) was stirred at room temperature for 16 hours. The reaction mixture was concentrated under vacuum. The crude product was purified by C18 reverse-phase column chromatography to obtain the title compound A1-12 (300 mg, yield: 38%).

[0266] MS(ESI): m / z = 398.2[M+H] + .

[0267] Step 12 2-Cyanoethyl((2R,3S,4R,5R)-2-((diethoxyphosphoryl)methoxy)-5-(2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)-4-(methoxy-d3)tetrahydrofuran-3-yl)diisopropylphosphoramidite A1 [ka] DIPEA (58 mg, 0.45 mmol) was added to a solution of compound A1-12 (100 mg, 0.252 mmol) in anhydrous dichloromethane (2 mL), followed by the addition of 3-[chloro-(diisopropylamino)phosphoryl]oxypropionitrile (83 mg, 0.35 mmol). The reaction mixture was stirred at room temperature for 2 hours and then quenched with MeOH. The reaction mixture was diluted with ethyl acetate and washed with saturated sodium bicarbonate, water, and brine. The organic layer was concentrated under vacuum. The crude product was purified by C18 reverse-phase column chromatography to obtain phosphoramidite monomer A1 (80 mg, yield: 53%).

[0268] MS(ESI): m / z = 596.0 [MH] + .

[0269] 1 H NMR (400 MHz, CD3OD)δ 7.74-7.70(m, 1H), 6.33-6.30(m, 1H), 5.84-5.82(m, 1H), 5.25-5.18(m, 1H), 4.51-4.44 (m, 1H), 4.27-3.67(m, 11H), 2.82-2.79(m, 2H), 1.41-1.36(m, 6H), 1.29-1.23(m, 12H).

[0270] 31 P NMR (400 MHz, CD3OD) δ 151.95, 150.57, 21.29.

[0271] Example 4: Synthesis of dsRNA containing deuterated chemical modification at the 5' end The synthesis of dsRNA was no different from that of a normal phosphoramidite solid-phase synthesis method. When synthesizing nucleotides modified at the end of the antisense strand, phosphoramidite monomer A1 synthesized in Example 3 was used at the corresponding position.

[0272] The synthesis process is briefly described below. Using a Dr. Oligo48 synthesizer (Biolytic), nucleoside phosphoramidite monomers were linked one by one using a synthesis program, starting with a Universal CPG vector. In addition to nucleoside phosphoramidite monomer A1 described in Example 3, other nucleoside monomer raw materials such as 2'-F RNA and 2'-O-methyl RNA were purchased from Shanghai Zhaowei or Suzhou Jima. By using 5-ethylthio-1H-tetrazole (ETT) as an activator (0.6 M acetonitrile solution) and a solution of 0.22 M PADS dissolved in acetonitrile and trimethylpyridine (Suzhou Kerama) in a volume ratio of 1:1 as a vulcanizing agent, a phosphorothioate can be introduced at the specified position. By using iodopyridine / aqueous solution (Kerama) as an oxidizing agent, an oxophosphate ester can be introduced at the specified position.

[0273] After solid-phase synthesis was completed, in some examples it was necessary to first remove the 5'-end protecting group with TMSI. For example, after solid-phase synthesis using phosphoramidite monomer A1, the crude oligoribonucleotide product was suspended in anhydrous acetonitrile, TMSI was added at room temperature, and after thorough stirring, the ethyl group (Et) in the oligoribonucleotide was removed to obtain oligoribonucleotide with NA0127. Next, the oligoribonucleotide was further dissolved from the solid support by immersion in a 3:1 28% aqueous ammonia and ethanol solution at 50°C for 16 hours. Then, it was centrifuged, the supernatant was transferred to another centrifuge tube, concentrated and evaporated, and then purified by C18 reverse-phase chromatography, removing DMTr with a mobile phase of 0.1 M TEAA and acetonitrile, and then with a 3% trifluoroacetic acid solution. The target oligonucleotide was collected, lyophilized, identified as the target product by LC-MS, and further quantified by UV (260 nm).

[0274] The obtained single-stranded oligonucleotides were paired complementaryally in equimolar ratios, annealed, and finally the resulting dsRNA was dissolved in 1×PBS and adjusted to the concentration required for the experiment for use.

[0275] The sense and antisense chain sequences modified with deuterated chemical modifications, 2'-fluoro groups, 2'-methoxy groups, etc., at the 5' end are shown in detail in Tables 1 and 2.

[0276] Example 5: Synthesis of dsRNA complex 1. Self-manufacturing of resin with carrier Compound NAG0052 (157 mg, 0.062 mmol), which contains a carboxylic acid group, was dissolved in anhydrous DMF (3 mL). After the substrate was completely dissolved, anhydrous acetonitrile (4 mL), DIEA (0.03 mL, 0.154 mmol, 2.5 eq), and HBTU (35 mg, 0.093 mmol, 1.5 eq) were added in sequence. After homogeneously mixing the reaction mixture, large-porous aminomethyl resin (476 mg, empty loading amount 0.41 mmol / g, target loading amount 0.1 mmol / g) was further added. The reaction mixture was placed in a shaker (temperature: 25°C, rotation speed: 200 rpm) and shaken overnight. The reaction mixture was filtered, and the filter cakes were washed sequentially with DCM and anhydrous acetonitrile, respectively. The solids were collected and dried under vacuum overnight.

[0277] The solid from the previous step was dispersed in anhydrous acetonitrile (5 mL), and pyridine (0.18 mL), DMAP (3 mg), NMI (0.12 mL), and CapB1 (2.68 mL) were added in sequence. The reaction mixture was shaken in a shaker (temperature: 25 °C, rotation speed: 200 rpm) for 2 hours. The reaction mixture was filtered, the filter cake was washed with anhydrous acetonitrile, the solid was collected, and dried overnight under vacuum to obtain a supported resin. The supported weight was measured to be 0.1 mmol / g.

[0278] 2. For NAG0052 already ligated to a resin, nucleoside monomers were ligated one by one from the 3'-5' direction according to the nucleotide sequence order, starting from the resin. The ligation to a single nucleoside monomer involves a four-step reaction: deprotection, coupling, capping, and oxidation or sulfidation. The procedure is common in this field.

[0279] Compound NAG0052 was linked to the sequence by solid-phase synthesis and then subjected to amine decomposition, after which some functional groups were removed from the structure of NAG0052 to obtain NAG0052'. 3. Using L96 instead of NAG0052, the related complex was synthesized according to the above steps. Here, L96 was obtained by preparation according to the method described in patent application WO2014025805A.

[0280] The produced dsRNA complexes had the sense and antisense strands shown in Tables 1 and 2.

[0281] The positive control dsRNA complex TRD007790 was prepared according to the method described in patent application WO2017059223A.

[0282] [Table 1-1]

[0283] [Table 1-2]

[0284] [Table 2-1]

[0285] [Table 2-2] JPEG2026520380000058.jpg110161

[0286] [Table 2-3]

[0287] Here, uppercase G, A, C, and U represent nucleotides containing guanine, adenine, cytosine, and uracil, respectively; lowercase m represents a 2'-methoxy group modification; lowercase f represents a 2'-fluoro modification; and lowercase s indicates that two nucleotides adjacent to the letter s are linked by a thiophosphodiester group. (-)hmpNA(A) is [ka] This represents, The structure of NAG0052' is, [ka] And, The structure of the L96' is [ka] can be, The structure of the NAG25s' is [ka] NA0127, NA0149, and InvdA represent the following, respectively: [ka] Here, the method for introducing NA0127 into dsRNA is as described in Example 4, the synthetic method for introducing NA0149 into dsRNA is referred to in reference US20190177729A, and the synthetic method for introducing InvdA into dsRNA is referred to in reference WO2017059223A.

[0288] [Table 3]

[0289] Example 6: Design of human LPA dsRNA Using human LPA (NM_005577.3) as the target gene, a 19 / 21nt dsRNA was designed to satisfy the general rules for active dsRNA. The sequences of the unmodified sense and antisense strands are shown in detail in Tables 4 and 5.

[0290] [Table 4]

[0291] [Table 5]

[0292] Example 7: On-target activity of dsRNA complex psiCHECK 9 concentration site In HEK293A cells, in vitro molecular-level simulations and on-target activity screening were performed on dsRNA / dsRNA complexes using nine concentration gradients.

[0293] HEK293A cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours prior to transfection, HEK293A cells were inoculated into 96-well plates at an inoculation density of 8 × 10⁴. 3 The cells were divided into wells, and the culture medium was 100 μL / well.

[0294] Cells were co-transfected with dsRNA complexes and corresponding plasmids using Lipofectamine 2000 (ThermoFisher, 11668019) according to the instructions for use, with 0.3 μL of Lipofectamine 2000 used per well. The plasmid transfection rate was 40 ng / well. For the on-target sequence plasmid, a total of nine concentration points were established for the dsRNA / dsRNA complex, with the highest concentration point being 20 nM. After 3-fold gradient dilution, the concentrations were 20 nM, 6.666666667 nM, 2.222222222 nM, 0.740740741 nM, 0.24691358 nM, 0.082304527 nM, 0.027434842 nM, 0.009144947 nM, and 0.003048316 nM. On-target levels were detected 24 hours after transfection using a Dual-Luciferase Reporter Assay System (Promega, E2940).

[0295] The results are shown in Table 6.

[0296] [Table 6-1]

[0297] [Table 6-2]

[0298] [Table 6-3]

[0299] Example 8. Inhibition of dsRNA on human LPA in human primary hepatocytes (PHH) - Inhibitory activity at 7 concentration points In human primary hepatocytes (PHH), PHH activity screening was performed for dsRNA sequences using seven concentration gradients. Each dsRNA sample had an initial final concentration of 20 nM after transfection, and consisted of 5-fold serial dilutions and seven concentration points.

[0300] PHH cells were cryopreserved in liquid nitrogen, and 24 hours before transfection, human primary hepatocytes (PHH) were revived and then inoculated into 96-well plates at an inoculation density of 3 × 10⁶. 4 The cells were 1 per well, and the culture medium was 80 μL per well.

[0301] Following the instructions for use of the product, dsRNA was transfected using Lipofectamine RNAi MAX (ThermoFisher, 13778150), and the final gradient concentrations of dsRNA transfection were 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, and 0.00128 nM. After 24 hours of processing, total cellular RNA was extracted using a high-throughput cell RNA extraction reagent kit, RNA reverse transcription experiments were performed, and quantitative real-time PCR detection was carried out to measure the mRNA level of human LPA. The mRNA level of human LPA was then corrected using the GAPDH internal reference gene level.

[0302] Here, during quantitative real-time PCR detection, probe Q-PCR detection experiments were used, and the primer information is shown in Tables 7 and 8.

[0303] After the Taqman probe Q-PCR detection experiment is completed, the corresponding Ct value is obtained according to a threshold automatically set in the system, and the expression of a certain gene can be quantified relatively by comparing the Ct values. Comparison of Ct refers to calculating the difference in gene expression by the difference value from the internal reference gene Ct value, and is also called 2-△△Ct, where △△Ct = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)].

[0304] The inhibition rate (%) was calculated as (1 - excess expression of the target gene) × 100%.

[0305] The results were shown as the excess percentage of human LPA mRNA expression in control dsRNA-treated cells. The IC50 results for inhibition rates are shown in Table 9.

[0306] The results showed that TJR100373 exhibited significantly superior activity compared to TJR100374-TJR100380, suggesting that the target gene fragment targeted by TJR100373 is more suitable as a target.

[0307] [Table 7]

[0308] [Table 8]

[0309] [Table 9]

[0310] Example 9: Measurement of in vivo activity of the dsRNA complex in humanized mouse (hu-Lp(a)). The humanized mouse (hu-Lp(a)) used in this embodiment was constructed by commissioning Shanghai Tuojie Biomedical Technology Co., Ltd. to Saiye (Suzhou) Biotechnology Co., Ltd.

[0311] The animals were divided into four equal groups based on their serum Lp(a) protein content, with six animals per group (two males and four females). Each group was administered physiological saline via subcutaneous injection. The positive control TRD007790 and the dsRNA complex TJR101079 of this disclosure were both administered at a dose of 3 mg / kg and a volume of 10 μL / g. On the day of administration, 40 μL of serum was collected before administration. The measured serum Lp(a) concentration was used as baseline data. The administration day was designated as day 1 (D1), and 40 μL of serum was collected each time on days 8 (D8), 29 (D29), 57 (D57), 85 (D85), and 99 (D99) after administration. The serum Lp(a) protein content was measured using an Abbott Ci4100 fully automated biochemical immunoassay system, and the inhibition of the dsRNA complex against the expression of humanized mouse (hu-Lp(a)) serum Lp(a) protein was calculated. The ratio of serum Lp(a) protein in each administration group to the blank control group after normalization was calculated, and the results are shown in Table 10. Each group was statistically analyzed against the blank administration group using one-way ANOVA.

[0312] As can be seen from the results in Table 10, no statistically significant difference was observed between the blank control group and the positive control group TRD007790 on day 99. However, TJR101079 still showed clear inhibition of Lp(a) protein expression, with a statistical significance of p<0.01. That is, TRD007790 did not show clear inhibitory activity on day 99, but TJR101079 still exhibited significant inhibitory activity.

[0313] This demonstrates that the dsRNA complex TJR101079 of this disclosure is excellent at long-term inhibiting Lp(a) protein expression.

[0314] [Table 10] In Table 10, * represents p ≤ 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001.

[0315] Example 10: Measurement of in vivo activity of the dsRNA complex in humanized mouse (hu-Lp(a)). The humanized mouse (hu-Lp(a)) used in this embodiment was constructed by commissioning Shanghai Tuojie Biomedical Technology Co., Ltd. to Saiye (Suzhou) Biotechnology Co., Ltd.

[0316] The mice were divided into four groups of six based on their serum Lp(a) protein content. Each group was administered subcutaneously, with dsRNA complexes TRD007790, TJR101079, TJR102134, and TJR102136 each administered at a dose of 1 mg / kg and a volume of 10 μL / g. 40 μL of serum was collected before administration on the day of administration, and the measured serum Lp(a) concentration was used as baseline data. The administration day was designated as day 1 (D1), and 40 μL of serum was collected again on day 28 (D28) after administration. The serum Lp(a) protein content was measured using an Abbott Ci4100 fully automated biochemical immunoassay system, and the inhibition of dsRNA complexes on humanized mouse (hu-Lp(a)) serum Lp(a) protein expression was calculated. The ratio of serum Lp(a) protein in each normalized treatment group to that of the blank control group was calculated, and the results are shown in Figures 1A, 1B, and 1C. The serum Lp(a) concentration on D28 in each group was compared with the serum Lp(a) before administration to obtain the excess serum Lp(a) ratio, and a statistical comparison was performed between TJR101079 and TRD007790, TJR102134, and TJR102136 using the paired t test.

[0317] As can be seen from the results in Figures 1A, 1B, and 1C, on day 28, the ratio of each group to pre-administration serum Lp(a) showed significant differences between TJR101079 and TRD007790, TJR102134, and TJR102136. TJR101079 to TRD007790 (*) p<0.05, TJR101079 to TJR102134 (*) p<0.05, and TJR101079 to TJR102136 (**) p<0.01.

[0318] This indicates that the long-term inhibitory effect of TJR101079 on Lp(a) protein expression is superior to that of the control groups TRD007790, TJR102134, and TJR102136. In other words, the dsRNA complex of this disclosure is significantly superior to the comparative complex in its long-term inhibitory effect on Lp(a) protein expression.

Claims

1. A double-stranded ribonucleic acid (dsRNA) that targets LPA, comprising a sense strand and an antisense strand forming a double-stranded region, wherein, The bare nucleotide sequence of the sense strand contains at least 17 consecutive nucleotides that differ from the nucleotide sequence of Sequence ID No. 1 by three or fewer nucleotides, and The bare nucleotide sequence of the antisense strand includes at least 19 consecutive nucleotides that differ from the nucleotide sequence of Sequence ID No. 2 by three or fewer nucleotides. Here, following the direction from the 5' end to the 3' end, The sense strand is a nucleotide in which the nucleotides at positions 7, 8, and 9 are modified with 2'-fluoro groups, and the nucleotides at the remaining positions are modified with 2'-methoxy groups. The antisense chain is a nucleotide modified with 2'-fluoro groups at positions 2 and 14, a nucleotide modified with 2'-methoxy groups or 2'-fluoro groups at positions 4, 6, 10, 12, 16 and 18 independently, and a nucleotide modified with 2'-methoxy groups at the remaining positions. The number of 2'-fluoromodified nucleotides in the antisense strand is 2 to 7. dsRNA.

2. The bare nucleotide sequence of the sense strand includes the nucleotide sequence shown in SEQ ID NO: 1, and the bare nucleotide sequence of the antisense strand includes the nucleotide sequence shown in SEQ ID NO:

2. The dsRNA according to claim 1.

3. The nucleotide at position 7 of the 5' end of the aforementioned antisense strand is a modified nucleotide, where, - The modified nucleotide is a nucleotide modified with a 2'-methoxy group, or - The modified nucleotide comprises the chemical modification shown in formula (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 Here, B represents the base at the position corresponding to the nucleotide at position 7 of the 5' end of the antisense strand. The dsRNA according to claim 1 or 2.

4. The nucleotide at position 1 of the 5' end of the aforementioned antisense strand is a modified nucleotide, where, - The modified nucleotide is a nucleotide modified with a 2'-methoxy group, or - The modified nucleotide is a chemically modified nucleotide shown in formula (II), 【Chemistry 2】 Here, B represents the base at the position corresponding to the nucleotide at position 1 of the 5' end of the antisense strand. The dsRNA according to any one of claims 1 to 3.

5. The aforementioned sense chain is 5’-N ａ N ａ N ａ N ａ N ａ N ａ N ｂ N ｂ N ｂ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ N ａ -3’ It includes the nucleotide sequence shown in the formula, Here, N ａ N is a nucleotide modified with a 2'-methoxy group, ｂ This is a nucleotide modified with 2'-fluoro. dsRNA according to any one of claims 1 to 4.

6. The aforementioned antisense chain is 5'-N ａ 'N ｂ 'N ａ 'X'N ａ 'X'N ａ 'N ａ 'N ａ 'X'N ａ 'X'N ａ 'N ｂ 'N ａ 'X'N ａ 'X'N ａ 'N ａ 'N ａ '-3', or 5'-N ａ 'N ｂ 'N ａ 'X'N ａ 'X'W'N ａ 'N ａ 'X'N ａ 'X'N ａ 'N ｂ 'N ａ 'X'N ａ 'X'N ａ 'N ａ 'N ａ '-3', or 5’-V’N ｂ ’N ａ ’X’N ａ ’X’N ａ ’N ａ ’N ａ ’X’N ａ ’X’N ａ ’N ｂ ’N ａ ’X’N ａ ’X’N ａ ’N ａ ’N ａ ’-3’ It includes the nucleotide sequence shown in the formula, Preferably, the antisense chain is 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’W’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-V’N ｂ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’、 5’-V’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ｂ ’N ａ ’N ｂ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’N ａ ’-3’ It includes the nucleotide sequence shown in the formula, Here, each X' is independently N ａ 'or N ｂ ' and N ａ ' is a nucleotide modified with a 2'-methoxy group, N ｂ ' represents a nucleotide modified with 2'-fluoro, W' represents a nucleotide containing the chemical modification shown in formulas (I), (I-1), (I-2) or a pharmaceutically acceptable salt thereof, and V' represents a chemically modified nucleotide shown in formula (II). dsRNA according to any one of claims 1 to 5.

7. At least one phosphodiester group in the sense chain and / or antisense chain is a phosphodiester group having a modifying group, preferably a thiophosphodiester group. dsRNA according to any one of claims 1 to 6.

8. The thiophosphodiester group is Between the first and second nucleotides at the 5' end of the sense strand, Between the second and third nucleotides at the 5' end of the sense strand, Between the first and second nucleotides at the 3' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, Between the second and third nucleotides at the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and It is located at at least one of the positions between the second and third nucleotides at the 3' end of the antisense strand, Preferably, the sense chain and / or antisense chain contains a plurality of thiophosphodiester groups, and the thiophosphodiester groups are Between the first and second nucleotides at the 5' end of the sense strand, Between the second and third nucleotides at the 5' end of the sense strand, Between the first and second nucleotides at the 3' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, and Between the second and third nucleotides at the 5' end of the antisense strand, and Between the first and second nucleotides at the 3' end of the antisense strand, It is located between the second and third nucleotides at the 3' end of the antisense strand, or The sense chain and / or antisense chain contains a plurality of thiophosphodiester groups, and the thiophosphodiester groups are Between the first and second nucleotides at the 5' end of the sense strand, Between the second and third nucleotides at the 5' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, and Between the second and third nucleotides at the 5' end of the antisense strand, and Between the first and second nucleotides at the 3' end of the antisense strand, Between the second and third nucleotides at the 3' end of the aforementioned antisense strand, The dsRNA according to claim 7.

9. The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 6, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NOs. 22 to 33, or SEQ ID NO: 35, or The sense strand includes the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NOs: 9 to 20 or SEQ ID NOs: 33 to 35. dsRNA according to any one of claims 1 to 8.

10. dsRNA complex, dsRNA according to any one of claims 1 to 9, and The dsRNA comprises a target ligand ligated to the terminal, Preferably, the target ligand is ligated to the 3' end of the sense strand of the dsRNA. dsRNA complex.

11. The target ligand comprises at least one target portion, The target portion is independently selected from galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, and N-isobutyryl-galactosamine. Preferably, the target portion is N-acetyl-galactosamine, More preferably, the target ligand comprises three identical or different target portions. The dsRNA complex according to claim 10.

12. The target ligand is a compound represented by formula (III-1) or (III-2) or a pharmaceutically acceptable salt thereof, where, The above formula (III-1) is, 【Transformation 3】 And, The above formula (III-2) is, 【Chemistry 4】 That is, The dsRNA complex according to claim 11.

13. The ligand is linked to the dsRNA via a phosphodiester group or a thiophosphodiester group, preferably via a phosphodiester group. The dsRNA complex according to any one of claims 10 to 12.

14. The sense strand includes the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NOs: 22 to 33, or SEQ ID NO: 35, or The sense strand includes the nucleotide sequence shown in SEQ ID NO: 4, and the antisense strand includes the nucleotide sequence shown in any one of SEQ ID NOs: 9 to 20, or SEQ ID NOs: 33 to 35, or The sense strand comprises the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand comprises the nucleotide sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 20, or SEQ ID NO:

33. The dsRNA complex according to any one of claims 10 to 13.

15. The dsRNA complex is selected from the following structures or pharmaceutically acceptable salts thereof: 【Transformation 5】 Here, Af = adenine 2'-F ribonucleoside, Cf = cytosine 2'-F ribonucleoside, Uf = Uracil 2'-F ribonucleoside, Gf = guanine 2'-F ribonucleoside, Am = adenine 2'-OMe ribonucleoside, Cm = cytosine 2'-OMe ribonucleoside, Gm = guanine 2'-OMe ribonucleoside, Um = uracil 2'-OMe ribonucleoside, 【Transformation 6】 This represents the thiophosphodiester group anionic form, 【Transformation 7】 This represents the phosphodiester group anion form, NAG0052' is, 【Transformation 8】 Representing, The dsRNA complex according to any one of claims 10 to 14.

16. A pharmaceutical composition, comprising the dsRNA described in any one of claims 1 to 9 and / or the dsRNA complex described in any one of claims 10 to 15, Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients. Pharmaceutical composition.

17. The use of a dsRNA according to any one of claims 1 to 9 and / or a dsRNA complex according to any one of claims 10 to 15 and / or a pharmaceutical composition according to claim 16 in the preparation of a drug, The aforementioned drugs are used to prevent and / or treat cardiovascular diseases, or The aforementioned drugs are used to prevent and / or treat diseases associated with elevated lipoprotein (a) and / or apolipoprotein (a) levels. Preferably, the disease associated with the elevated levels of lipoprotein (a) and / or apolipoprotein (a) is selected from cardiovascular diseases. The aforementioned cardiovascular diseases are selected from ischemic stroke, atherosclerosis, thrombosis, coronary heart disease, lower extremity artery disease, aortic valve stenosis, myocardial infarction, coronary artery stenosis, carotid artery stenosis, femoral artery stenosis, and heart failure. use.

18. A method for inhibiting LPA expression, comprising administering an effective amount or effective dose of the dsRNA described in any one of claims 1 to 9 and / or the dsRNA complex described in any one of claims 10 to 15 and / or the pharmaceutical composition described in claim 16 to the target. method.

19. A method for in vivo delivery of dsRNA to the liver to inhibit LPA expression and / or replication, comprising administering an effective amount or effective dose of the dsRNA according to any one of claims 1 to 9 and / or the dsRNA complex according to any one of claims 10 to 15 and / or the pharmaceutical composition according to claim 16 to the target. method.

20. A dsRNA according to any one of claims 1 to 9 and / or a dsRNA complex according to any one of claims 10 to 15 and / or a pharmaceutical composition according to claim 16. cell.

21. A dsRNA according to any one of claims 1 to 9 and / or a dsRNA complex according to any one of claims 10 to 15 and / or a pharmaceutical composition according to claim 16. Reagent kit.

22. A method for preparing dsRNA, dsRNA complexes, or pharmaceutical compositions, Synthesizing a dsRNA according to any one of claims 1 to 9 and / or a dsRNA complex according to any one of claims 10 to 15, and / or The preparation of the pharmaceutical composition described in claim 16, method.

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